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Mapping of the ATP-binding Sites on Inositol 1,4,5-Trisphosphate Receptor Type 1 and Type 3 Homotetramers by Controlled Proteolysis and Photoaffinity Labeling

Mapping of the ATP-binding Sites on Inositol 1,4,5-Trisphosphate Receptor Type 1 and Type 3... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 5, Issue of February 2, pp. 3492–3497, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Mapping of the ATP-binding Sites on Inositol 1,4,5-Trisphosphate Receptor Type 1 and Type 3 Homotetramers by Controlled Proteolysis and Photoaffinity Labeling* Received for publication, July 11, 2000, and in revised form, October 11, 2000 Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M006082200 Karlien Maes‡§, Ludwig Missiaen‡, Jan B. Parys‡¶, Patrick De Smet‡i, Ilse Sienaert‡i, Etienne Waelkens**, Geert Callewaert‡, and Humbert De Smedt‡ From the ‡Laboratorium voor Fysiologie and the **Laboratorium voor Biochemie, K.U.Leuven Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium Submillimolar ATP concentrations strongly enhance rameric structures (9 –12). The different IP R isoforms show )-induced Ca re- functional differences in their regulation by IP and by several the inositol 1,4,5-trisphosphate (IP 3 3 lease, by binding specifically to ATP-binding sites on the modulators of IP -induced Ca release (13–15). receptor (IP R). To locate those ATP-binding sites on IP ATP regulates the IP R in a concentration-dependent man- 3 3 R1 and IP R3, both proteins were expressed in Sf9 IP 3 3 ner: Submillimolar concentrations enhance IP -induced Ca a- insect cells and covalently labeled with 8-azido-[ release (16 –20), whereas millimolar levels of ATP inhibit IP - P]ATP. IP R1 and IP R3 were then purified and sub- 3 3 induced Ca release by competing with IP for the IP -binding 3 3 jected to a controlled proteolysis, and the labeled pro- site (18 –23). The stimulatory effect of ATP is likely to occur via teolytic fragments were identified by site-specific binding to one or more sites on the IP R, because purified IP Rs 3 3 R1 were labeled, each antibodies. Two fragments of IP 32 bind [a- P]ATP in a specific manner (17, 20, 24). The number containing one of the previously proposed ATP-binding and the localization of these sites have, however, not yet been sites with amino acid sequence GXGXXG (amino acids determined. Based on the glycine-rich amino acid sequence R3, only 1773–1780 and 2016 –2021, respectively). In IP GXGXXG (25), two ATP-binding sites were postulated on the one fragment was labeled. This fragment contained the neuronal form of IP R1 (aa 1773–1780 and 2016 –2021). The GXGXXG sequence (amino acids 1920 –1925), which is former is only present in IP R1, whereas the latter is common R isoforms. The presence of conserved in the three IP to the three IP R isoforms (2, 26 –28). In a previous study, we multiple interaction sites for ATP was also evident from have expressed the cDNA domains of IP R1 containing these -induced Ca release in permeabilized A7r5 3 the IP glycine-rich motifs as glutathione S-transferase (GST) fusion cells, which depended on ATP over a very broad concen- proteins in bacteria and showed that they both were able to tration range from micromolar to millimolar. bind ATP (29). The aim of the present study was to determine the location and number of ATP-binding sites on the intact IP R. We did this by photoaffinity labeling with 8-azido-[a- Inositol 1,4,5-trisphosphate (IP ) is an intracellular second P]ATP of microsomes of Sf9 insect cells expressing recombi- messenger that mediates the release of Ca from internal nant IP R1 or IP R3 homotetramers, followed by purification 3 3 stores by binding to the IP receptor (IP R), an intracellular 3 3 and controlled proteolysis with chymotrypsin. We found that Ca -release channel (1). The IP R is composed of three func- controlled proteolysis of IP R1 and IP R3 yielded roughly the 3 3 tionally different domains: an N-terminal IP -binding region, a same major fragments, indicating that both isoforms have a large transducing domain, and a C-terminal channel region (2). similar general structure. Moreover, the results indicated that The transducing domain contains interaction sites for several 21 21 the IP R1 contained two ATP-binding sites, because two sepa- modulators of IP -induced Ca release such as Ca , calmod- 3 rate fragments obtained by proteolysis were labeled. These two ulin, kinases, phosphatases, ATP, and FKBP12 (reviewed in fragments each contained one of the presumed ATP-binding Refs. 1 and 3). IP Rs are encoded by three different genes, sites (aa 1773–1780 and 2016 –2021, respectively), as identified resulting in the existence of IP R1, IP R2, and IP R3, and the 3 3 3 using site-specific antibodies. In IP R3, only one proteolytic various IP R isoforms are distributed in a tissue-specific man- 3 fragment was labeled. This fragment contained the proposed ner (4 –7). Nearly all cell types coexpress at least two IP R ATP-binding site that is conserved in all IP R isoforms (aa isoforms (4, 5, 8), which are mostly co-organized in heterotet- 3 1920 –1925). The unequal number of ATP-binding sites in IP R1 and IP R3 could have implications for the modulation of 3 3 these isoforms by ATP. Recently, we found that IP R1 and * This work was supported in part by Grant 99/08 of the Concerted Actions, by Grant P4/23 of the Interuniversity Poles of Attraction Pro- IP R3 have a different ATP affinity (EC values of 1.6 and 177 3 50 gram of the Belgian State, and by Grants 3.0207.99 and G.0322.97 of mM, respectively) (24). In this study, the ATP dependence of the Foundation for Scientific Research-Flanders (FWO). The costs of IP -induced Ca release measured in permeabilized A7r5 publication of this article were defrayed in part by the payment of page cells, which express both IP R1 and IP R3, extended over a charges. This article must therefore be hereby marked “advertisement” 3 3 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. very broad range from micromolar to millimolar concentra- A Research Associate of the FWO. tions. This finding confirms the presence of multiple nucleo- i Senior Research Assistants of the FWO. tide-binding sites with different affinities for ATP in IP R1 and § To whom correspondence should be addressed: Tel.: 32-16-345-736; IP R3. Fax: 32-16-345-991; E-mail: [email protected]. 3 The abbreviations used are: IP , inositol 1,4,5-trisphosphate; IP R, 3 3 EXPERIMENTAL PROCEDURES IP receptor; aa, amino acids; GST, glutathione S-transferase; CHAPS, Materials—CHAPS was obtained from Pierce (Rockford, IL). Chymo- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis. trypsin, N-tosyl-L-phenylalanine chloromethyl ketone, heparin-agar- 3492 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. ATP-binding Sites on IP R 3493 ose, and N-acetyl-D-glucosamine were from Sigma Chemical Co. (St. lack of a known cell type that abundantly expresses this iso- Louis, MO). 8-Azido-[a- P]ATP (2 mCi/ml, 12 Ci/mmol) was purchased form. We therefore expressed IP R1 or IP R3 in Sf9 insect cells, 45 3 3 from ICN Pharmaceuticals Inc. (Costa Mesa, CA). CaCl (2.2 mCi/ml, resulting in a 2.5 times higher expression of IP R1 and a .50 mgofCa 134 /ml), wheat germ agglutinin-Sepharose 6MB, Rainbow times higher expression of IP R3 as compared with rabbit molecular mass markers, the anti-mouse and anti-rabbit alkaline phos- cerebellar and 16HBE14o-microsomes, respectively (24, 30). In phatase-coupled secondary antibodies, and the Vistra ECF substrate were from Amersham Pharmacia Biotech AB (Uppsala, Sweden). the purification procedures described by Chadwick et al. (39) Expression of IP R1 and IP R3 in Insect Sf9 cells—The full-length 3 3 and Parys et al. (32), microsomes were first solubilized by a mouse IP R1 and the full-length rat IP R3 were expressed in insect Sf9 3 3 detergent followed by chromatography on heparin- and lectin- cells as described by Sipma et al. (30) and by Maes et al. (24), based matrices. This method was based on the ability of hep- respectively. arin to bind to the IP R with high affinity (41– 43) and on the a- Photoaffinity Labeling with 8-Azido-[ P]ATP of Recombinant 3 presence of N-glycosylation sites on 2 asparagine residues pres- IP R—Microsomes of Sf9 insect cells were prepared as described (31). Photoaffinity labeling of microsomes containing either IP3R1 or IP R3 3 ent in IP R1 (44). It has been suggested that IP R3 is also a 3 3 a- with 8-azido-[ P]ATP was performed exactly as described in Maes et glycoprotein (45), although only one N-glycosylation site is al. (24). predicted based on the primary sequence (46). Purification of Recombinant IP R1 and IP R3 from Sf9 Micro- 3 3 In this study, we have used an identical approach to purify somes—The purification of IP Rs was based on the method described by recombinant IP R1 and IP R3 overexpressed in Sf9 insect cells. Parys et al. (32). Microsomes of Sf9 cells expressing IP R1 and IP R3 in 3 3 3 3 a concentration of 10 mg of protein/ml were centrifuged, and the pellet It had to be verified whether the glycosylation patterns of these was solubilized (at 5 mg/ml) for 1.5 h at 4 °C in buffer A (50 m proteins were the same as in mammalian cells. Briefly, micro- Tris-HCl, pH 7.4, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, somes from Sf9 cells overexpressing either IP R1 or IP R3 3 3 0.83 mM benzamidine, and 10 mM 2-mercaptoethanol) with addition of were solubilized with 2.5% CHAPS. Subsequently, the solubi- 200 mM NaCl, 77 nM aprotinin, 1.1 mM leupeptin, 0.7 mM pepstatin A, lized microsomes were incubated with heparin-agarose. After 2.5% CHAPS, and 1% L-a-phosphatidylcholine. After centrifugation, the supernatant was diluted with an equal volume of buffer A with elution of the bound fraction, the latter was incubated with addition of 400 mM NaCl. The diluted supernatant was incubated for 30 wheat germ agglutinin-Sepharose. Both receptors could be pu- min with heparin-agarose beads (112.5 ml/mg of protein). The eluate rified with high efficiency and were recognized by isoform- obtained in buffer A with 600 mM NaCl, 0.75% CHAPS, and 0.3% specific antibodies (Fig. 1, A and B, first lane of each blot), L-a-phosphatidylcholine, was incubated for 2 h with wheat germ agglu- confirming that they are both glycoproteins and that the post- tinin-Sepharose (75 ml/mg of protein). After wash steps in high (600 mM) and low (100 mM) salt conditions, the specifically bound proteins were translational glycosylation of the IP Rs in insect cells is similar eluted in low salt conditions with 300 mM N-acetyl-D-glucosamine. All to that in mammalian cells. The purified IP R1 migrated on centrifugation steps were for 17 min at 35,700 3 g at 4 °C. SDS-PAGE with a molecular mass of 273 kDa, which deviated Controlled Proteolysis—Purified IP R was partially digested with from the molecular mass of 313 kDa predicted from the pri- mg/ml) for 2, 5, 10, or 30 min on ice as described chymotrypsin (0.05 mary structure (Fig. 1A, first lane of each blot). The purified previously (32). The digestion was stopped by the addition of 100 mg/ml IP R3 also migrated with a lower apparent molecular mass N-tosyl-L-phenylalanine chloromethyl ketone and by boiling the sam- ples for 5 min in sample buffer for SDS-PAGE. (248 kDa) than predicted (304 kDa) (Fig. 1B, first lane of each Antibodies and Western Blotting—The polyclonal antibody against blot). Because a similar behavior is also found for endogenous the C terminus of mouse IP R1 (Rbt03), the mouse monoclonal antibody IP Rs from, e.g. cerebellar or 16HBE14o-cells (20, 47), this against an N-terminal epitope of human IP R3 (MMAtype3) (Transduc- discrepancy is likely due to aberrant mobility of higher molec- tion Laboratories, Lexington, KY) and the polyclonal antibody against ular mass proteins on SDS-PAGE. the Ca -binding domain cytI3b (amino acids 378 – 450) in the IP - Controlled Proteolysis and Identification of Proteolytic Frag- binding domain of mouse IP R1 (33) were characterized earlier (4, 30, 34). A novel antibody was raised against the luminal Ca -binding ments—The purified IP Rs were subjected to a controlled pro- fragment LoopI17a of mouse IP R1 (aa 2463–2528) (35). Two rabbits teolysis with chymotrypsin (0.05 mg/ml, up to 30 min on ice), were injected subcutaneously and intramuscularly with Freund’s com- and the digestion fragments were detected by a panel of differ- plete adjuvant containing 0.5 mg of LoopI17a fused to GST. Animals ent site-specific antibodies (Fig. 1, A and B, and Table I). No were boosted 2 weeks later with the same antigen in Freund’s incom- degradation of the intact IP R was observed during incubation plete adjuvant and regularly thereafter. After three boost injections, 3 both rabbits produced high titers of antibody. Both antibodies (named without chymotrypsin (Fig. 1, first lane of each blot). anti-loopI17a-1 and anti-loopI17a-2) reacted with mouse, rat, human, For IP R1, four site-specific antibodies were used, of which hamster, and rabbit IP R1. They also recognized rat IP R3, although 3 3 the epitopes were spread over the whole sequence. The anti- A polyclonal antibody directed against residues with lower sensitivity. cytI3b-2 antibody (30) is directed against a Ca -binding site in 1829 –1848 of human IP R1 was purchased from Alexis Corp. the IP -binding domain (33). The anti-(1829 –1848) antibody (La ¨ ufelfingen, Switzerland). A polyclonal antibody against the C termi- (Alexis Corp.) recognized an amino acid stretch (residues nus of human IP R3 was from Santa Cruz Biotechnology (Santa Cruz, CA). The various microsomal preparations were analyzed on 3–12% 1829 –1848) located in the regulatory domain between the two Laemmli-type gels and transferred to Immobilon-P (Millipore Corp., putative ATP-binding sites (residues 1773–1780 and 2016 – Bedford, MA). Immunodetection of the proteins on the transfers was 2021) (2, 26 –28). A third antibody, anti-loopI17a-2, was raised exactly as described previously (30, 36). 45 21 21 against the luminal Ca -binding fragment (35). Finally, Ca Fluxes—IP -induced Ca release from permeabilized A7r5 Rbt03 (30, 34) recognized the C terminus of IP R1. The proteo- monolayers was described elsewhere (16). The added ATP concentra- 3 tions are indicated in the legend to Fig. 4. The medium for the challenge lytic pattern, resulting from up to 30 min of incubation with with IP contained 120 mM KCl, 30 mM imidazole-HCl, pH 6.8, and 1 chymotrypsin, and as detected by the four antibodies against M EGTA. IP R1, is shown in Fig. 1A. We determined the length of the fragments using Rainbow molecular mass markers. Based on RESULTS AND DISCUSSION these data, we were able to localize the chymotrypsin-sensitive Purification of IP Rs from Sf9 Insect Cells—To allow an sites on IP R1 (Fig. 2A). The sum of the molecular mass of the accurate analysis involving controlled proteolysis and immu- five major proteolytic fragments (40, 65, 80, 40, and 90 kDa) nostaining, purification of the IP R is needed. IP R1 has been 3 3 was close to the molecular mass of the intact IP R1 (313 kDa). purified from cerebellum (37, 38), smooth muscle (39, 40) and This result was in complete agreement with the study of oocytes (32). Until now, no IP R3 has been purified due to the Yoshikawa et al. (48), where trypsin was used to digest cere- bellum-purified IP R1 and where five similar major proteoly- J. B. Parys, unpublished data. sis-insensitive fragments were found. Although we were able to ATP-binding Sites on IP 3494 R digestion was performed for 30 min. Particularly, a 145-kDa fragment, precursor of the 65- and 80-kDa fragments should be recognized by the anti-cytI3b antibody and a 185-kDa frag- ment, precursor of the successive fragments of 65, 80, and 40 kDa should be recognized by the anti-(1829 –1848) antibody. Because it is possible that these intermediate fragments have a short life time, we decreased the time of proteolysis to 2, 5, and 10 min, respectively (Fig. 1A, insets of blots 1 and 2). Upon staining with the anti-cytI3b-2 antibody (blot 1 and inset), we detected a proteolytic band corresponding to a molecular mass of 145 kDa, which is most intense at 2 and 5 min of incubation with chymotrypsin. This fragment is rapidly degraded into smaller fragments, because it is poorly or not visible in the proteolytic patterns representing 10 and 30 min of incubation of IP R1 with chymotrypsin. Upon staining with the anti- (1829 –1848) antibody, no clear fragment with a mass of 185 kDa was visible, even at shorter time points (blot 2 and inset). Because the corresponding predicted fragment was also not detected in the study of Yoshikawa et al. (48), it is conceivable that the latter intermediate fragment is rapidly degraded into smaller subfragments during proteolysis and has there- fore a steady-state level below the detection limit for the anti- bodies. All identified fragments, with indication of their molec- ular mass and the recognizing antibodies, are represented in Table II. The same type of experiment was performed for IP R3. Only two site-specific antibodies are available for this isoform: the MMAtype3 antibody (Transduction Laboratories) (4) directed against the N terminus, and the anti-CIII antibody (Santa Cruz Biotechnologies) against the C terminus. However, the anti-loopI17a-2 antibody could also recognize IP R3, although with lower sensitivity. The proteolytic pattern as stained by the three antibodies against IP R3 is shown in Fig. 1B. The time dependence of the occurrence of the proteolytic fragments was also investigated for IP R3, but incubation with chymotrypsin for shorter times (2–10 min) revealed the same pattern of proteolytic fragments (data not shown). All identified frag- ments, with their molecular mass and the recognizing antibod- ies, are represented in Table III. In addition we have verified the N-terminal boundaries of some of the major proteolytic fragments by N-terminal amino acid microsequencing (data not FIG.1. Controlled proteolysis of purified IP R1 (A) and IP R3 3 3 shown). A schematic presentation of IP R3 with the major (B). IP Rs were purified from 1 mg of Sf9 microsomes as described proteolytic fragments (105, 70, 35, and 95 kDa) is shown in Fig. under “Experimental Procedures.” The duration of the controlled pro- 2B. The sum of the molecular mass of the fragments was close teolysis with chymotrypsin (0.05 mg/ml on ice) is indicated at the top of each lane. Control IP Rs were not treated with chymotrypsin. The to the molecular mass of the intact receptor (304 kDa) as proteins were separated by SDS-PAGE and transferred to Immo- calculated from the cloned rat IP R3. The general structure of bilon-P. The blots were probed with site-specific antibodies: In A, blot 1, IP R3 resembled that of IP R1: Both receptor isoforms were 3 3 anti-cytI3b-2 (dilution 1/300); blot 2, anti-(1829 –1848) (dilution 1/700); sensitive to proteolysis at similar sites. Only the chymotrypsin- blot 3, anti-loopI17a-2 (dilution 1/1000); and blot 4, Rbt03 (dilution 1/10000). In B, blot 1, MMAtype3 (dilution 1/1000); blot 2, anti- sensitive site that is present in the IP -binding domain of loopI17a-2 (dilution 1/200); and blot 3, anti-CIII (dilution 1/250). Posi- IP R1, could not be detected in IP R3. This could however be 3 3 tions of the molecular mass markers (in kDa) are indicated. due to the lack of an antibody that recognized the relevant part of the IP -binding domain. Alternatively, it is also possible that TABLE I 3 Overview of antibodies IP R3 lacks the chymotrypsin-sensitive site in the IP -binding 3 3 domain. It is conceivable that the proteolysis-sensitive sites IP R a Antibody Epitope References/source subtype represent regions that are exposed on the surface of the protein and thereby accessible to the proteolytic enzymes as well as to Anti-cytI3b-2 1 m378–450 (30) Anti-(1829–1848) 1 h1829–1848 Alexis Corp. different modulators of IP -induced Ca release. Because Anti-loopI17a-2 1 m2463–2528 This study functional IP Rs are mostly organized in heterotetramers (9 – 3 r2391–2456 12), it can be expected that corresponding regions of the differ- Rbt03 1 m2735–2749 (30, 34) ent IP R isoforms are exposed at the surface of the receptor MMAtype3 3 h22–230 (4) 3 Anti-CIII 3 h2652–2671 Santa Cruz Biotechnologies protein so that they can be properly regulated. a Identification of Photoaffinity-labeled Proteolytic Frag- m, mouse; h, human; r, rabbit. ments—In a previous study, we showed that two GST fusion recognize most of the intermediate digestion products with proteins, each containing a putative ATP-binding domain of site-specific antibodies, two proteolytic fragments, which were IP R1, could bind ATP (29). Both predicted ATP-binding do- predicted based on Fig. 2A, could not be detected when the mains were situated near chymotrypsin-sensitive sites (Fig. ATP-binding Sites on IP R 3495 FIG.2. Schematic representation of the proteolytic fragments of IP R1 (A) and IP R3 (B). The top line indicates the molecular mass and 3 3 the number of amino acids of the IP R. The horizontal bar represents a scheme of the IP R with the chymotrypsin-sensitive sites (indicated by the 3 3 scissors), the size of the proteolytic fragments (in kDa), the proposed ATP-binding sites, and the epitopes of the site-specific antibodies. TABLE II TABLE III Identification of proteolytic fragments of IP R1 by site-specific Identification of proteolytic fragments of IP R3 by site-specific 3 3 32 32 antibodies and 8-azido-[a- P]ATP labeling antibodies and 8-azido-[a- P]ATP labeling IP R1 was purified from Sf9 insect cells and subjected to a controlled IP R3 was purified from Sf9 insect cells and subjected to a controlled 3 3 proteolysis. The proteolytic fragments were identified with site-specific proteolysis. The proteolytic fragments were identified with site-specific antibodies. The proteolytic fragments (represented in kDa), obtained antibodies. Proteolytic fragments (represented in kDa) recognized by a after 2–30 min of incubation with chymotrypsin and recognized by a particular antibody are indicated by an “x”. “x ” indicates that the used particular antibody are indicated by an “x.” “x ” indicates that the used antibody detected the particular fragment with a very weak intensity. antibody detected the particular fragment with a very weak intensity, The same method was used for IP R3, which was labeled with 8-azido- 32 32 or that the fragment was very weakly labeled with 8-azido-[a- P]ATP. [a- P]ATP prior to purification and controlled proteolysis. As illus- The same method was used for IP R1, which was labeled with 8-azido- trated in Fig. 2B, the smallest labeled fragment was the 95-kDa [a- P]ATP prior to purification and controlled proteolysis. The pres- C-terminal fragment. The details of the photoaffinity labeling, purifi- ence of two different labeled sites (on fragments of 90 and 40 kDa, cation, and controlled proteolysis are described under “Experimental respectively) follows from their specific identification with different Procedures.” antibodies (Fig. 2A). The details of the photoaffinity labeling, purifica- kDa MMAtype3 Anti-loopI17a-2 Anti-CIII P label tion, and controlled proteolysis are described under “Experimental Procedures.” 248 x x x x 32 210 x kDa Anti-cytI3B-2 Anti-(1829–1848) Anti-loopI17a-2 Rbt03 P label 200 x x x 273 x x x x x 175 x 225 x x x 130 x xx 210 x x x x 105 x 185 x x 95 x x x 145 x 130 x x x x 120 x x labeled proteolytic fragments by site-specific antibodies. The 105 x 90 x x x two smallest labeled proteolytic fragments of IP R1 (90 and 40 65 x kDa, Fig. 3A) were recognized by the Rbt03 antibody and the 40 x x anti-(1829 –1848) antibody, respectively (Table II), indicating that they represented the proteolytic fragments containing the 2A). It is therefore likely that they are both accessible to ATP in previously proposed ATP-binding sites (Fig. 2A). the intact protein. To prove this, we incubated microsomes IP R3 contained only one of these proposed ATP-binding from Sf9 cells expressing recombinant IP R1 with the photoaf- sites, which is conserved in all IP R isoforms and which is also 3 3 finity label 8-azido-[a- P]ATP. Covalent labeling of the ATP- located near a chymotrypsin-sensitive site (Fig. 2B). To confirm this, we performed the same photoaffinity labeling experiment binding sites by UV irradiation was followed by purification and controlled proteolysis of IP R1 and identification of the for the IP R3 isoform. The smallest labeled band of 95 kDa 3 3 ATP-binding Sites on IP 3496 R FIG.3. Photoaffinity labeling of IP R1 (A) and IP R3 (B) fol- 3 3 lowed by controlled proteolysis. Microsomes from Sf9 insect cells were incubated with 20 mM 8-azido-[a- P]ATP and subsequently irra- diated with UV light for 2.5 min. IP R1 and IP R3 were purified from 3 3 those microsomes after solubilization of the microsomes with CHAPS and binding to heparin-agarose and subsequently wheat germ aggluti- nin-Sepharose. IP Rs were digested with 0.05 mg/ml chymotrypsin for 30 min on ice (lanes 2) or were not treated with chymotrypsin (lanes 1). FIG.4. ATP dependence of IP -induced Ca release. Nonmito- After SDS-PAGE and blotting, labeled IP Rs were visualized using the chondrial Ca stores in permeabilized A7r5 cells, loaded to steady Storm 840 PhosphorImager (Molecular Dynamics). Positions of the 45 21 state with Ca , were incubated in efflux medium for 10 min, at which molecular mass markers (in kDa) are indicated. The details of the time 1 mM IP plus the indicated ATP concentration was added for 2 photoaffinity labeling and the proteolysis are described under “Exper- min. The stimulation of the Ca release by ATP is expressed as the imental Procedures.” percentage increase in the Ca release above the control value, ob- tained in the absence of ATP. Data are the means of four independent experiments. The error bars smaller than the data symbol are not (Fig. 3B) was recognized by the anti-CIII antibody (Table III), indicated. indicating that this band represented the proteolytic fragment containing the putative ATP-binding site of IP R3 (Fig. 2B). In ent data do not allow a determination of whether the presence summary, covalent labeling with 8-azido-[a- P]ATP occurred of two IP R isoforms in A7r5 is reflected in two separate stim- at two different proteolytic fragments of IP R1 and only at one ulatory ATP-binding sites. However, for preparations from rat proteolytic fragment of IP R3. The labeled fragments contained cerebellum containing nearly exclusively IP R1, the maximum the two previously proposed ATP-binding sites, one of which is stimulation by ATP was found at 50 mM.At1mM ATP, IP - conserved in all IP R isoforms. induced Ca release in cerebellar preparations was close to The unequal number of ATP-binding sites found in IP R1 control values (50), whereas 1 mM ATP was the maximum and IP R3 may explain the differential modulation of these stimulatory concentration in A7r5 cells. The much higher max- isoforms by ATP. IP R1 showed a higher affinity for ATP than imum for ATP stimulation found for A7r5 cells is therefore very IP R3 (13, 14, 24), suggesting that the upstream ATP-binding probably a reflection of the presence of IP R3. It was also not site, which is only present in IP R1, is a high-affinity binding possible to decide whether these properties of IP R3 are in- site. Moreover, IP R3 displayed a broader nucleotide specificity ferred in A7r5 cells by homo- or heterotetramers. Coimmuno- than IP R1 (24), because it bound equally well ATP and GTP. precipitation experiments indicated that a significant fraction The latter property can be assigned to the ATP-binding site of IP R1 and IP R3 expressed in A7r5 cells is present as het- 3 3 present in IP R3 and conserved in all IP R isoforms. The ATP- 3 3 erotetramers. Our data clearly showed that the presence of binding site that is only present in IP R1 was more specific for different ATP-binding sites on IP R1 and IP R3 resulted in a 3 3 adenine nucleotides like ATP and ADP (24). nucleotide sensitivity of IP -induced Ca release that ex- 21 3 ATP Dependence of IP -induced Ca Release—In permeabi- tended over a broad concentration range. The ATP concentra- lized A7r5 cells, which express IP R1 and IP R3ina3to1 ratio 3 3 tion that yielded maximum stimulation seems very variable (5), ATP dependence of IP -induced Ca release was found and to be dependent on the IP R isoform composition in the over a very broad concentration range (Fig. 4). ATP stimulated particular cell type. IP -induced Ca release from the low micromolar range up to 1mM. At still higher ATP concentrations, the release was Acknowledgments—We thank Lea Bauwens, Jerry Renders, Luce Heremans, Anja Florizoone, Marina Crabbe ´ , Hilde Van Weijenbergh, inhibited probably due to competition of ATP for the IP -bind- Ire ` ne Willems, Yves Parijs, and Raphael Verbist for their skillful tech- ing site (18 –23). The broad concentration dependence in A7r5 nical assistance. We acknowledge the generous gifts of the p400C1 cells is in very good agreement with the different ATP affinities plasmid containing the IP R1 cDNA by Drs. K. Mikoshiba and A. described previously for recombinant IP R1 and IP R3 (EC Miyawaki (University of Tokyo, Japan) and the pCB61 plasmid con- 3 3 50 taining the IP R3 cDNA by Dr. G. I. Bell (Howard Hughes Medical values of 1.6 mM and 177 mM, respectively) (24). This difference 3 Institute, University of Chicago, IL). We thank Drs. S. Joseph (Thomas in ATP affinities between IP R1 and IP R3 was also observed 3 3 Jefferson University School of Medicine, Philadelphia, PA) and G. by other groups: recent findings of Hagar and Ehrlich (49) Guillemette (University of Sherbrooke, Quebec, Canada) for the kind demonstrated that IP R3 incorporated in lipid bilayers was 3 gift of anti-IP R3 antibodies used in some control experiments. activated by ATP with an EC of about 3 mM, whereas a much REFERENCES lower EC (40 mM) was observed for IP R1 (19). Moreover, IP - 50 3 3 1. Berridge, M. J. (1993) Nature 361, 315–325 induced Ca release in genetically engineered DT40 B cells that 2. Mignery, G. 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Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N., and Mikoshiba, K. (1994) Receptors and Channels 2, 9 –22 Mikoshiba, K. (1989) Nature 342, 32–38 47. Sienaert, I., Huyghe, S., Parys, J. B., Malfait, M., Kunzelmann, K., De Smedt, 27. Mignery, G. A., Newton, C. L., Archer, B. T., III, and Su ¨ dhof, T. C. (1990) H., Verleden, G. M., and Missiaen, L. (1998) Pflu ¨ gers Arch. 436, 40–48 J. Biol. Chem. 265, 12679 –12685 48. Yoshikawa, F., Iwasaki, H., Michikawa, T., Furuichi, T., and Mikoshiba, K. 28. Ferris, C. D., and Snyder, S. H. (1992) Annu. Rev. Physiol. 54, 469 – 488 (1999) J. Biol. Chem. 274, 316 –327 29. Maes, K., Missiaen, L., Parys, J. B., Sienaert, I., Bultynck, G., Zizi, M., De Smet, P., Casteels, R., and De Smedt, H. (1999) Cell Calcium 25, 49. Hagar, R. E., and Ehrlich, B. E. (2000) Biophys. J. 79, 271–278 50. Kaplin, A. I., Snyder, S. H., and Linden, D. J. (1996) J. Neurosci. 16, 143–152 30. Sipma, H., De Smet, P., Sienaert, I., Vanlingen, S., Missiaen, L., Parys, J. B., 2002–2011 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Mapping of the ATP-binding Sites on Inositol 1,4,5-Trisphosphate Receptor Type 1 and Type 3 Homotetramers by Controlled Proteolysis and Photoaffinity Labeling

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 5, Issue of February 2, pp. 3492–3497, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Mapping of the ATP-binding Sites on Inositol 1,4,5-Trisphosphate Receptor Type 1 and Type 3 Homotetramers by Controlled Proteolysis and Photoaffinity Labeling* Received for publication, July 11, 2000, and in revised form, October 11, 2000 Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M006082200 Karlien Maes‡§, Ludwig Missiaen‡, Jan B. Parys‡¶, Patrick De Smet‡i, Ilse Sienaert‡i, Etienne Waelkens**, Geert Callewaert‡, and Humbert De Smedt‡ From the ‡Laboratorium voor Fysiologie and the **Laboratorium voor Biochemie, K.U.Leuven Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium Submillimolar ATP concentrations strongly enhance rameric structures (9 –12). The different IP R isoforms show )-induced Ca re- functional differences in their regulation by IP and by several the inositol 1,4,5-trisphosphate (IP 3 3 lease, by binding specifically to ATP-binding sites on the modulators of IP -induced Ca release (13–15). receptor (IP R). To locate those ATP-binding sites on IP ATP regulates the IP R in a concentration-dependent man- 3 3 R1 and IP R3, both proteins were expressed in Sf9 IP 3 3 ner: Submillimolar concentrations enhance IP -induced Ca a- insect cells and covalently labeled with 8-azido-[ release (16 –20), whereas millimolar levels of ATP inhibit IP - P]ATP. IP R1 and IP R3 were then purified and sub- 3 3 induced Ca release by competing with IP for the IP -binding 3 3 jected to a controlled proteolysis, and the labeled pro- site (18 –23). The stimulatory effect of ATP is likely to occur via teolytic fragments were identified by site-specific binding to one or more sites on the IP R, because purified IP Rs 3 3 R1 were labeled, each antibodies. Two fragments of IP 32 bind [a- P]ATP in a specific manner (17, 20, 24). The number containing one of the previously proposed ATP-binding and the localization of these sites have, however, not yet been sites with amino acid sequence GXGXXG (amino acids determined. Based on the glycine-rich amino acid sequence R3, only 1773–1780 and 2016 –2021, respectively). In IP GXGXXG (25), two ATP-binding sites were postulated on the one fragment was labeled. This fragment contained the neuronal form of IP R1 (aa 1773–1780 and 2016 –2021). The GXGXXG sequence (amino acids 1920 –1925), which is former is only present in IP R1, whereas the latter is common R isoforms. The presence of conserved in the three IP to the three IP R isoforms (2, 26 –28). In a previous study, we multiple interaction sites for ATP was also evident from have expressed the cDNA domains of IP R1 containing these -induced Ca release in permeabilized A7r5 3 the IP glycine-rich motifs as glutathione S-transferase (GST) fusion cells, which depended on ATP over a very broad concen- proteins in bacteria and showed that they both were able to tration range from micromolar to millimolar. bind ATP (29). The aim of the present study was to determine the location and number of ATP-binding sites on the intact IP R. We did this by photoaffinity labeling with 8-azido-[a- Inositol 1,4,5-trisphosphate (IP ) is an intracellular second P]ATP of microsomes of Sf9 insect cells expressing recombi- messenger that mediates the release of Ca from internal nant IP R1 or IP R3 homotetramers, followed by purification 3 3 stores by binding to the IP receptor (IP R), an intracellular 3 3 and controlled proteolysis with chymotrypsin. We found that Ca -release channel (1). The IP R is composed of three func- controlled proteolysis of IP R1 and IP R3 yielded roughly the 3 3 tionally different domains: an N-terminal IP -binding region, a same major fragments, indicating that both isoforms have a large transducing domain, and a C-terminal channel region (2). similar general structure. Moreover, the results indicated that The transducing domain contains interaction sites for several 21 21 the IP R1 contained two ATP-binding sites, because two sepa- modulators of IP -induced Ca release such as Ca , calmod- 3 rate fragments obtained by proteolysis were labeled. These two ulin, kinases, phosphatases, ATP, and FKBP12 (reviewed in fragments each contained one of the presumed ATP-binding Refs. 1 and 3). IP Rs are encoded by three different genes, sites (aa 1773–1780 and 2016 –2021, respectively), as identified resulting in the existence of IP R1, IP R2, and IP R3, and the 3 3 3 using site-specific antibodies. In IP R3, only one proteolytic various IP R isoforms are distributed in a tissue-specific man- 3 fragment was labeled. This fragment contained the proposed ner (4 –7). Nearly all cell types coexpress at least two IP R ATP-binding site that is conserved in all IP R isoforms (aa isoforms (4, 5, 8), which are mostly co-organized in heterotet- 3 1920 –1925). The unequal number of ATP-binding sites in IP R1 and IP R3 could have implications for the modulation of 3 3 these isoforms by ATP. Recently, we found that IP R1 and * This work was supported in part by Grant 99/08 of the Concerted Actions, by Grant P4/23 of the Interuniversity Poles of Attraction Pro- IP R3 have a different ATP affinity (EC values of 1.6 and 177 3 50 gram of the Belgian State, and by Grants 3.0207.99 and G.0322.97 of mM, respectively) (24). In this study, the ATP dependence of the Foundation for Scientific Research-Flanders (FWO). The costs of IP -induced Ca release measured in permeabilized A7r5 publication of this article were defrayed in part by the payment of page cells, which express both IP R1 and IP R3, extended over a charges. This article must therefore be hereby marked “advertisement” 3 3 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. very broad range from micromolar to millimolar concentra- A Research Associate of the FWO. tions. This finding confirms the presence of multiple nucleo- i Senior Research Assistants of the FWO. tide-binding sites with different affinities for ATP in IP R1 and § To whom correspondence should be addressed: Tel.: 32-16-345-736; IP R3. Fax: 32-16-345-991; E-mail: [email protected]. 3 The abbreviations used are: IP , inositol 1,4,5-trisphosphate; IP R, 3 3 EXPERIMENTAL PROCEDURES IP receptor; aa, amino acids; GST, glutathione S-transferase; CHAPS, Materials—CHAPS was obtained from Pierce (Rockford, IL). Chymo- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis. trypsin, N-tosyl-L-phenylalanine chloromethyl ketone, heparin-agar- 3492 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. ATP-binding Sites on IP R 3493 ose, and N-acetyl-D-glucosamine were from Sigma Chemical Co. (St. lack of a known cell type that abundantly expresses this iso- Louis, MO). 8-Azido-[a- P]ATP (2 mCi/ml, 12 Ci/mmol) was purchased form. We therefore expressed IP R1 or IP R3 in Sf9 insect cells, 45 3 3 from ICN Pharmaceuticals Inc. (Costa Mesa, CA). CaCl (2.2 mCi/ml, resulting in a 2.5 times higher expression of IP R1 and a .50 mgofCa 134 /ml), wheat germ agglutinin-Sepharose 6MB, Rainbow times higher expression of IP R3 as compared with rabbit molecular mass markers, the anti-mouse and anti-rabbit alkaline phos- cerebellar and 16HBE14o-microsomes, respectively (24, 30). In phatase-coupled secondary antibodies, and the Vistra ECF substrate were from Amersham Pharmacia Biotech AB (Uppsala, Sweden). the purification procedures described by Chadwick et al. (39) Expression of IP R1 and IP R3 in Insect Sf9 cells—The full-length 3 3 and Parys et al. (32), microsomes were first solubilized by a mouse IP R1 and the full-length rat IP R3 were expressed in insect Sf9 3 3 detergent followed by chromatography on heparin- and lectin- cells as described by Sipma et al. (30) and by Maes et al. (24), based matrices. This method was based on the ability of hep- respectively. arin to bind to the IP R with high affinity (41– 43) and on the a- Photoaffinity Labeling with 8-Azido-[ P]ATP of Recombinant 3 presence of N-glycosylation sites on 2 asparagine residues pres- IP R—Microsomes of Sf9 insect cells were prepared as described (31). Photoaffinity labeling of microsomes containing either IP3R1 or IP R3 3 ent in IP R1 (44). It has been suggested that IP R3 is also a 3 3 a- with 8-azido-[ P]ATP was performed exactly as described in Maes et glycoprotein (45), although only one N-glycosylation site is al. (24). predicted based on the primary sequence (46). Purification of Recombinant IP R1 and IP R3 from Sf9 Micro- 3 3 In this study, we have used an identical approach to purify somes—The purification of IP Rs was based on the method described by recombinant IP R1 and IP R3 overexpressed in Sf9 insect cells. Parys et al. (32). Microsomes of Sf9 cells expressing IP R1 and IP R3 in 3 3 3 3 a concentration of 10 mg of protein/ml were centrifuged, and the pellet It had to be verified whether the glycosylation patterns of these was solubilized (at 5 mg/ml) for 1.5 h at 4 °C in buffer A (50 m proteins were the same as in mammalian cells. Briefly, micro- Tris-HCl, pH 7.4, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, somes from Sf9 cells overexpressing either IP R1 or IP R3 3 3 0.83 mM benzamidine, and 10 mM 2-mercaptoethanol) with addition of were solubilized with 2.5% CHAPS. Subsequently, the solubi- 200 mM NaCl, 77 nM aprotinin, 1.1 mM leupeptin, 0.7 mM pepstatin A, lized microsomes were incubated with heparin-agarose. After 2.5% CHAPS, and 1% L-a-phosphatidylcholine. After centrifugation, the supernatant was diluted with an equal volume of buffer A with elution of the bound fraction, the latter was incubated with addition of 400 mM NaCl. The diluted supernatant was incubated for 30 wheat germ agglutinin-Sepharose. Both receptors could be pu- min with heparin-agarose beads (112.5 ml/mg of protein). The eluate rified with high efficiency and were recognized by isoform- obtained in buffer A with 600 mM NaCl, 0.75% CHAPS, and 0.3% specific antibodies (Fig. 1, A and B, first lane of each blot), L-a-phosphatidylcholine, was incubated for 2 h with wheat germ agglu- confirming that they are both glycoproteins and that the post- tinin-Sepharose (75 ml/mg of protein). After wash steps in high (600 mM) and low (100 mM) salt conditions, the specifically bound proteins were translational glycosylation of the IP Rs in insect cells is similar eluted in low salt conditions with 300 mM N-acetyl-D-glucosamine. All to that in mammalian cells. The purified IP R1 migrated on centrifugation steps were for 17 min at 35,700 3 g at 4 °C. SDS-PAGE with a molecular mass of 273 kDa, which deviated Controlled Proteolysis—Purified IP R was partially digested with from the molecular mass of 313 kDa predicted from the pri- mg/ml) for 2, 5, 10, or 30 min on ice as described chymotrypsin (0.05 mary structure (Fig. 1A, first lane of each blot). The purified previously (32). The digestion was stopped by the addition of 100 mg/ml IP R3 also migrated with a lower apparent molecular mass N-tosyl-L-phenylalanine chloromethyl ketone and by boiling the sam- ples for 5 min in sample buffer for SDS-PAGE. (248 kDa) than predicted (304 kDa) (Fig. 1B, first lane of each Antibodies and Western Blotting—The polyclonal antibody against blot). Because a similar behavior is also found for endogenous the C terminus of mouse IP R1 (Rbt03), the mouse monoclonal antibody IP Rs from, e.g. cerebellar or 16HBE14o-cells (20, 47), this against an N-terminal epitope of human IP R3 (MMAtype3) (Transduc- discrepancy is likely due to aberrant mobility of higher molec- tion Laboratories, Lexington, KY) and the polyclonal antibody against ular mass proteins on SDS-PAGE. the Ca -binding domain cytI3b (amino acids 378 – 450) in the IP - Controlled Proteolysis and Identification of Proteolytic Frag- binding domain of mouse IP R1 (33) were characterized earlier (4, 30, 34). A novel antibody was raised against the luminal Ca -binding ments—The purified IP Rs were subjected to a controlled pro- fragment LoopI17a of mouse IP R1 (aa 2463–2528) (35). Two rabbits teolysis with chymotrypsin (0.05 mg/ml, up to 30 min on ice), were injected subcutaneously and intramuscularly with Freund’s com- and the digestion fragments were detected by a panel of differ- plete adjuvant containing 0.5 mg of LoopI17a fused to GST. Animals ent site-specific antibodies (Fig. 1, A and B, and Table I). No were boosted 2 weeks later with the same antigen in Freund’s incom- degradation of the intact IP R was observed during incubation plete adjuvant and regularly thereafter. After three boost injections, 3 both rabbits produced high titers of antibody. Both antibodies (named without chymotrypsin (Fig. 1, first lane of each blot). anti-loopI17a-1 and anti-loopI17a-2) reacted with mouse, rat, human, For IP R1, four site-specific antibodies were used, of which hamster, and rabbit IP R1. They also recognized rat IP R3, although 3 3 the epitopes were spread over the whole sequence. The anti- A polyclonal antibody directed against residues with lower sensitivity. cytI3b-2 antibody (30) is directed against a Ca -binding site in 1829 –1848 of human IP R1 was purchased from Alexis Corp. the IP -binding domain (33). The anti-(1829 –1848) antibody (La ¨ ufelfingen, Switzerland). A polyclonal antibody against the C termi- (Alexis Corp.) recognized an amino acid stretch (residues nus of human IP R3 was from Santa Cruz Biotechnology (Santa Cruz, CA). The various microsomal preparations were analyzed on 3–12% 1829 –1848) located in the regulatory domain between the two Laemmli-type gels and transferred to Immobilon-P (Millipore Corp., putative ATP-binding sites (residues 1773–1780 and 2016 – Bedford, MA). Immunodetection of the proteins on the transfers was 2021) (2, 26 –28). A third antibody, anti-loopI17a-2, was raised exactly as described previously (30, 36). 45 21 21 against the luminal Ca -binding fragment (35). Finally, Ca Fluxes—IP -induced Ca release from permeabilized A7r5 Rbt03 (30, 34) recognized the C terminus of IP R1. The proteo- monolayers was described elsewhere (16). The added ATP concentra- 3 tions are indicated in the legend to Fig. 4. The medium for the challenge lytic pattern, resulting from up to 30 min of incubation with with IP contained 120 mM KCl, 30 mM imidazole-HCl, pH 6.8, and 1 chymotrypsin, and as detected by the four antibodies against M EGTA. IP R1, is shown in Fig. 1A. We determined the length of the fragments using Rainbow molecular mass markers. Based on RESULTS AND DISCUSSION these data, we were able to localize the chymotrypsin-sensitive Purification of IP Rs from Sf9 Insect Cells—To allow an sites on IP R1 (Fig. 2A). The sum of the molecular mass of the accurate analysis involving controlled proteolysis and immu- five major proteolytic fragments (40, 65, 80, 40, and 90 kDa) nostaining, purification of the IP R is needed. IP R1 has been 3 3 was close to the molecular mass of the intact IP R1 (313 kDa). purified from cerebellum (37, 38), smooth muscle (39, 40) and This result was in complete agreement with the study of oocytes (32). Until now, no IP R3 has been purified due to the Yoshikawa et al. (48), where trypsin was used to digest cere- bellum-purified IP R1 and where five similar major proteoly- J. B. Parys, unpublished data. sis-insensitive fragments were found. Although we were able to ATP-binding Sites on IP 3494 R digestion was performed for 30 min. Particularly, a 145-kDa fragment, precursor of the 65- and 80-kDa fragments should be recognized by the anti-cytI3b antibody and a 185-kDa frag- ment, precursor of the successive fragments of 65, 80, and 40 kDa should be recognized by the anti-(1829 –1848) antibody. Because it is possible that these intermediate fragments have a short life time, we decreased the time of proteolysis to 2, 5, and 10 min, respectively (Fig. 1A, insets of blots 1 and 2). Upon staining with the anti-cytI3b-2 antibody (blot 1 and inset), we detected a proteolytic band corresponding to a molecular mass of 145 kDa, which is most intense at 2 and 5 min of incubation with chymotrypsin. This fragment is rapidly degraded into smaller fragments, because it is poorly or not visible in the proteolytic patterns representing 10 and 30 min of incubation of IP R1 with chymotrypsin. Upon staining with the anti- (1829 –1848) antibody, no clear fragment with a mass of 185 kDa was visible, even at shorter time points (blot 2 and inset). Because the corresponding predicted fragment was also not detected in the study of Yoshikawa et al. (48), it is conceivable that the latter intermediate fragment is rapidly degraded into smaller subfragments during proteolysis and has there- fore a steady-state level below the detection limit for the anti- bodies. All identified fragments, with indication of their molec- ular mass and the recognizing antibodies, are represented in Table II. The same type of experiment was performed for IP R3. Only two site-specific antibodies are available for this isoform: the MMAtype3 antibody (Transduction Laboratories) (4) directed against the N terminus, and the anti-CIII antibody (Santa Cruz Biotechnologies) against the C terminus. However, the anti-loopI17a-2 antibody could also recognize IP R3, although with lower sensitivity. The proteolytic pattern as stained by the three antibodies against IP R3 is shown in Fig. 1B. The time dependence of the occurrence of the proteolytic fragments was also investigated for IP R3, but incubation with chymotrypsin for shorter times (2–10 min) revealed the same pattern of proteolytic fragments (data not shown). All identified frag- ments, with their molecular mass and the recognizing antibod- ies, are represented in Table III. In addition we have verified the N-terminal boundaries of some of the major proteolytic fragments by N-terminal amino acid microsequencing (data not FIG.1. Controlled proteolysis of purified IP R1 (A) and IP R3 3 3 shown). A schematic presentation of IP R3 with the major (B). IP Rs were purified from 1 mg of Sf9 microsomes as described proteolytic fragments (105, 70, 35, and 95 kDa) is shown in Fig. under “Experimental Procedures.” The duration of the controlled pro- 2B. The sum of the molecular mass of the fragments was close teolysis with chymotrypsin (0.05 mg/ml on ice) is indicated at the top of each lane. Control IP Rs were not treated with chymotrypsin. The to the molecular mass of the intact receptor (304 kDa) as proteins were separated by SDS-PAGE and transferred to Immo- calculated from the cloned rat IP R3. The general structure of bilon-P. The blots were probed with site-specific antibodies: In A, blot 1, IP R3 resembled that of IP R1: Both receptor isoforms were 3 3 anti-cytI3b-2 (dilution 1/300); blot 2, anti-(1829 –1848) (dilution 1/700); sensitive to proteolysis at similar sites. Only the chymotrypsin- blot 3, anti-loopI17a-2 (dilution 1/1000); and blot 4, Rbt03 (dilution 1/10000). In B, blot 1, MMAtype3 (dilution 1/1000); blot 2, anti- sensitive site that is present in the IP -binding domain of loopI17a-2 (dilution 1/200); and blot 3, anti-CIII (dilution 1/250). Posi- IP R1, could not be detected in IP R3. This could however be 3 3 tions of the molecular mass markers (in kDa) are indicated. due to the lack of an antibody that recognized the relevant part of the IP -binding domain. Alternatively, it is also possible that TABLE I 3 Overview of antibodies IP R3 lacks the chymotrypsin-sensitive site in the IP -binding 3 3 domain. It is conceivable that the proteolysis-sensitive sites IP R a Antibody Epitope References/source subtype represent regions that are exposed on the surface of the protein and thereby accessible to the proteolytic enzymes as well as to Anti-cytI3b-2 1 m378–450 (30) Anti-(1829–1848) 1 h1829–1848 Alexis Corp. different modulators of IP -induced Ca release. Because Anti-loopI17a-2 1 m2463–2528 This study functional IP Rs are mostly organized in heterotetramers (9 – 3 r2391–2456 12), it can be expected that corresponding regions of the differ- Rbt03 1 m2735–2749 (30, 34) ent IP R isoforms are exposed at the surface of the receptor MMAtype3 3 h22–230 (4) 3 Anti-CIII 3 h2652–2671 Santa Cruz Biotechnologies protein so that they can be properly regulated. a Identification of Photoaffinity-labeled Proteolytic Frag- m, mouse; h, human; r, rabbit. ments—In a previous study, we showed that two GST fusion recognize most of the intermediate digestion products with proteins, each containing a putative ATP-binding domain of site-specific antibodies, two proteolytic fragments, which were IP R1, could bind ATP (29). Both predicted ATP-binding do- predicted based on Fig. 2A, could not be detected when the mains were situated near chymotrypsin-sensitive sites (Fig. ATP-binding Sites on IP R 3495 FIG.2. Schematic representation of the proteolytic fragments of IP R1 (A) and IP R3 (B). The top line indicates the molecular mass and 3 3 the number of amino acids of the IP R. The horizontal bar represents a scheme of the IP R with the chymotrypsin-sensitive sites (indicated by the 3 3 scissors), the size of the proteolytic fragments (in kDa), the proposed ATP-binding sites, and the epitopes of the site-specific antibodies. TABLE II TABLE III Identification of proteolytic fragments of IP R1 by site-specific Identification of proteolytic fragments of IP R3 by site-specific 3 3 32 32 antibodies and 8-azido-[a- P]ATP labeling antibodies and 8-azido-[a- P]ATP labeling IP R1 was purified from Sf9 insect cells and subjected to a controlled IP R3 was purified from Sf9 insect cells and subjected to a controlled 3 3 proteolysis. The proteolytic fragments were identified with site-specific proteolysis. The proteolytic fragments were identified with site-specific antibodies. The proteolytic fragments (represented in kDa), obtained antibodies. Proteolytic fragments (represented in kDa) recognized by a after 2–30 min of incubation with chymotrypsin and recognized by a particular antibody are indicated by an “x”. “x ” indicates that the used particular antibody are indicated by an “x.” “x ” indicates that the used antibody detected the particular fragment with a very weak intensity. antibody detected the particular fragment with a very weak intensity, The same method was used for IP R3, which was labeled with 8-azido- 32 32 or that the fragment was very weakly labeled with 8-azido-[a- P]ATP. [a- P]ATP prior to purification and controlled proteolysis. As illus- The same method was used for IP R1, which was labeled with 8-azido- trated in Fig. 2B, the smallest labeled fragment was the 95-kDa [a- P]ATP prior to purification and controlled proteolysis. The pres- C-terminal fragment. The details of the photoaffinity labeling, purifi- ence of two different labeled sites (on fragments of 90 and 40 kDa, cation, and controlled proteolysis are described under “Experimental respectively) follows from their specific identification with different Procedures.” antibodies (Fig. 2A). The details of the photoaffinity labeling, purifica- kDa MMAtype3 Anti-loopI17a-2 Anti-CIII P label tion, and controlled proteolysis are described under “Experimental Procedures.” 248 x x x x 32 210 x kDa Anti-cytI3B-2 Anti-(1829–1848) Anti-loopI17a-2 Rbt03 P label 200 x x x 273 x x x x x 175 x 225 x x x 130 x xx 210 x x x x 105 x 185 x x 95 x x x 145 x 130 x x x x 120 x x labeled proteolytic fragments by site-specific antibodies. The 105 x 90 x x x two smallest labeled proteolytic fragments of IP R1 (90 and 40 65 x kDa, Fig. 3A) were recognized by the Rbt03 antibody and the 40 x x anti-(1829 –1848) antibody, respectively (Table II), indicating that they represented the proteolytic fragments containing the 2A). It is therefore likely that they are both accessible to ATP in previously proposed ATP-binding sites (Fig. 2A). the intact protein. To prove this, we incubated microsomes IP R3 contained only one of these proposed ATP-binding from Sf9 cells expressing recombinant IP R1 with the photoaf- sites, which is conserved in all IP R isoforms and which is also 3 3 finity label 8-azido-[a- P]ATP. Covalent labeling of the ATP- located near a chymotrypsin-sensitive site (Fig. 2B). To confirm this, we performed the same photoaffinity labeling experiment binding sites by UV irradiation was followed by purification and controlled proteolysis of IP R1 and identification of the for the IP R3 isoform. The smallest labeled band of 95 kDa 3 3 ATP-binding Sites on IP 3496 R FIG.3. Photoaffinity labeling of IP R1 (A) and IP R3 (B) fol- 3 3 lowed by controlled proteolysis. Microsomes from Sf9 insect cells were incubated with 20 mM 8-azido-[a- P]ATP and subsequently irra- diated with UV light for 2.5 min. IP R1 and IP R3 were purified from 3 3 those microsomes after solubilization of the microsomes with CHAPS and binding to heparin-agarose and subsequently wheat germ aggluti- nin-Sepharose. IP Rs were digested with 0.05 mg/ml chymotrypsin for 30 min on ice (lanes 2) or were not treated with chymotrypsin (lanes 1). FIG.4. ATP dependence of IP -induced Ca release. Nonmito- After SDS-PAGE and blotting, labeled IP Rs were visualized using the chondrial Ca stores in permeabilized A7r5 cells, loaded to steady Storm 840 PhosphorImager (Molecular Dynamics). Positions of the 45 21 state with Ca , were incubated in efflux medium for 10 min, at which molecular mass markers (in kDa) are indicated. The details of the time 1 mM IP plus the indicated ATP concentration was added for 2 photoaffinity labeling and the proteolysis are described under “Exper- min. The stimulation of the Ca release by ATP is expressed as the imental Procedures.” percentage increase in the Ca release above the control value, ob- tained in the absence of ATP. Data are the means of four independent experiments. The error bars smaller than the data symbol are not (Fig. 3B) was recognized by the anti-CIII antibody (Table III), indicated. indicating that this band represented the proteolytic fragment containing the putative ATP-binding site of IP R3 (Fig. 2B). In ent data do not allow a determination of whether the presence summary, covalent labeling with 8-azido-[a- P]ATP occurred of two IP R isoforms in A7r5 is reflected in two separate stim- at two different proteolytic fragments of IP R1 and only at one ulatory ATP-binding sites. However, for preparations from rat proteolytic fragment of IP R3. The labeled fragments contained cerebellum containing nearly exclusively IP R1, the maximum the two previously proposed ATP-binding sites, one of which is stimulation by ATP was found at 50 mM.At1mM ATP, IP - conserved in all IP R isoforms. induced Ca release in cerebellar preparations was close to The unequal number of ATP-binding sites found in IP R1 control values (50), whereas 1 mM ATP was the maximum and IP R3 may explain the differential modulation of these stimulatory concentration in A7r5 cells. The much higher max- isoforms by ATP. IP R1 showed a higher affinity for ATP than imum for ATP stimulation found for A7r5 cells is therefore very IP R3 (13, 14, 24), suggesting that the upstream ATP-binding probably a reflection of the presence of IP R3. It was also not site, which is only present in IP R1, is a high-affinity binding possible to decide whether these properties of IP R3 are in- site. Moreover, IP R3 displayed a broader nucleotide specificity ferred in A7r5 cells by homo- or heterotetramers. Coimmuno- than IP R1 (24), because it bound equally well ATP and GTP. precipitation experiments indicated that a significant fraction The latter property can be assigned to the ATP-binding site of IP R1 and IP R3 expressed in A7r5 cells is present as het- 3 3 present in IP R3 and conserved in all IP R isoforms. The ATP- 3 3 erotetramers. Our data clearly showed that the presence of binding site that is only present in IP R1 was more specific for different ATP-binding sites on IP R1 and IP R3 resulted in a 3 3 adenine nucleotides like ATP and ADP (24). nucleotide sensitivity of IP -induced Ca release that ex- 21 3 ATP Dependence of IP -induced Ca Release—In permeabi- tended over a broad concentration range. The ATP concentra- lized A7r5 cells, which express IP R1 and IP R3ina3to1 ratio 3 3 tion that yielded maximum stimulation seems very variable (5), ATP dependence of IP -induced Ca release was found and to be dependent on the IP R isoform composition in the over a very broad concentration range (Fig. 4). ATP stimulated particular cell type. IP -induced Ca release from the low micromolar range up to 1mM. At still higher ATP concentrations, the release was Acknowledgments—We thank Lea Bauwens, Jerry Renders, Luce Heremans, Anja Florizoone, Marina Crabbe ´ , Hilde Van Weijenbergh, inhibited probably due to competition of ATP for the IP -bind- Ire ` ne Willems, Yves Parijs, and Raphael Verbist for their skillful tech- ing site (18 –23). The broad concentration dependence in A7r5 nical assistance. We acknowledge the generous gifts of the p400C1 cells is in very good agreement with the different ATP affinities plasmid containing the IP R1 cDNA by Drs. K. 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Sipma, H., De Smet, P., Sienaert, I., Vanlingen, S., Missiaen, L., Parys, J. B., 2002–2011

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Published: Feb 1, 2001

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